Stabilized adducts of menadione bisulfite with p-aminobenzoic acid or adenine

ABSTRACT

Stabilized adducts of K vitaminic compounds, as menadione bisulfite or a derivative thereof and stabilizing vitamins as, particularly, nicotine amide or nicotinic acid. Feed compositions containing the same. Process for their preparation.

This application is a continuation of application Ser. No. 273,379,filed June 15, 1981 abandoned, which is a continuation of applicationSer. No. 102,445 filed Dec. 11, 1979 now abandoned, which is acontinuation in part application of Ser. No. 971,031 filed Dec. 19,1978, now abandoned.

This invention relates to K vitaminic compounds and stabilizing vitaminadducts, consistent with K vitaminic compounds, as well as to thestabilized adducts thus obtained.

It relates also to the preparation of such adducts, particularly, toadducts stabilized with nicotine amide or nicotinic acid.

In another aspect, the present invention relates to composition foranimal feed, containing the same as additives, as well as compositionsfor use in human therapy.

Particularly, the present invention relates to novel adducts ofmenadione bisulfite with compounds exhibiting vitaminic activity, suchas for example, nicotinic acid and nicotine amide.

Adducts of menadione bisulfite have long been known, such as adducts ofmenadione sodium bisulfite (vitamin K₃). See U.S. Pat. No. 2,367,302.These adducts are water-soluble and have the substantialantihaemorrhagic activity of the K vitamin type and are not an irritantas menadione itself.

Menadione sodium bisulfite has been already widely used as an additivefor feeds (particularly for poultry) and is also useful in the field ofhuman therapy.

However, it suffers from the disadvantages of a limited stability tolight, moisture, heat, and, particularly in a solution, having pH valueshigher than neutrality. Thus, the antihaemorrhagic activity of menadioneor vitamin K₃, comprising the active principle thereof, will deterioratein feed premixtures with the passage of time, and the productscontaining it do not stand up well in extended storage under unfavorableconditions.

In fact, it was shown (Baker et al J.A.C.S. 64 1096, 1942) that even ata relatively low pH (lower than neutrality), isomerization occursresulting in a product which has no antihaemorrhagic activity. The rateof isomerization increases according to the pH of the medium, and at apH of about 8.5, the adduct decomposes, releasing menadione.

Since feed premixtures may be moist and have a pH higher thanneutrality, attempts were made to obviate the resulting unstability insuch products by synthetizing the less water-soluble adducts ofmenadione bisulfite, not having the disadvantages of menadione sodiumbisulfite. That is, they crystalized with 2-3 water molecules (thuspromoting some solubility), and had a pH of 6.5 in saturated aqueoussolutions, which is sufficiently high to promote a rather high rate ofisomerization and accordingly a reduction in vitaminic activity.

Research in this direction resulted in the preparation of adducts ofmenadione bisulfite, wherein sodium was replaced by a slightly basicorganic compound, particularly dicyanodiamidine, 2,4,6-triamino-1,3,5-triazine and pyrimidine substituted at position 2 (U.S. Pat. No.3,328,169). These adducts had the characteristics supposedly useful fora stable menadione bisulfite apt to be introduced in feed premixtures(or for other uses), namely:

(1) absence of crystallization water

(2) low water-solubility; and

(3) pH of saturated solutions preferably lower than about 4.5.

The most accepted among these adducts is menadione pyrimidinolebisulfite (menadione-bisulfite-2-hydroxy-4,6-dimethyl-pyrimidine)(MPB).

In this adduct the amount of menadione is 45.63% by weight, and theamount of dimethyl pyrimidinole is 32.63% by weight. It is supposed thatthe basic portion of the MPB molecule enhances the K₃ vitaminicactivity. However, it suffers from two disadvantages. First, althoughMPB is a pyrimidinic compound similar to natural pyrimidines, which aregenerally metabolized by an organism, it is not known how this syntheticproduct is metabolized. Second, its high proportion in the finalproduct, in which it performs only the function of a stabilizer, leavesa high percentage of the product "inert" with respect to therapeuticactivity.

Therefore, the present invention, on the one hand, eliminates the highproportion of an inert component, and, on the other hand, introduces afurther component having a useful vitaminic activity which adds to thatof the K vitaminic compound.

The products according to the present invention are adducts of menadionebisulfite with compounds, also having vitaminic activity, and at thesame time having stabilizing properties with respect to the former.These adducts are the products of an addition reaction, producing anactual vitaminic unit or complex having a combined activity, and thusoffering both the economic advantage of reduced cost due to the absenceof an inert component possessing no vitaminic activity and the technicaladvantage, a surprisingly more active product. Thus, the presentinvention provides a unique product which combines a plurality of usefulactivities with an accompanying reduction in volume and handlingotherwise required for obtaining the same results.

The products, according to the present invention, are obtained byreacting menadione sodium bisulfite or other highly water-solublebisulfite adducts, such as potassium or ammonium adducts, with salts ofe.g., nicotine amide or nicotinic acid in the presence of strong mineralacids, such as hydrochloric acid, sulphuric acid and phosphoric acid, orcertain organic acids, such as acetic acid.

Preferably, the adducts are prepared by mixing concentrated solutions ofmenadione alkaline bisulfite with concentrated solutions of a salt ofnicotine amide or nicotinic acid, such as chlorohydrate or sulphate,thus obtaining a precipitate which is easily separated by filtration.

Menadione itself could also be reacted with the bisulfite salt ofnicotine amide or nicotinic acid; however, the reaction rate would belower, and thus, the process is not preferred.

The adducts of menadione bisulfite with nicotine amide and nicotinicacid, as obtained according to the present invention, are free of waterof crystallization and are slightly soluble in water and in an aqueoussolution having a pH lower than 4.5 (maximum 3.0).

Because of their decomposition at a pH 8.5 in menadione and nicotineamide or nicotinic acid (sodium salt), the percentage of menadione isdetermined by the method shown in USA Pharmacopeia, 15th Edition, page394.

The most preferred adduct of menadione bisulfite with nicotine amide hasthe general formula C₁₇ H₁₆ O₆ N₂ S, and a molecular weight of 376.42.It consists of:

Menadione: 45.74% by weight

Nicotine amide: 32.44% by weight

Its IR spectrum is shown in FIG. 1 (wave number in abscissa, (cm⁻¹); inordinate: transmittance (%)).

The most preferred adduct of menadione bisulfite with nicotinic acid hasthe general formula C₁₇ H₁₅ O₇ NS, and a molecular weight of 377.36. Itconsists of:

Menadione: 45.62% by weight

Nicotinic acid: 32.62% by weight

Its IR spectrum is shown in FIG. 2 (wave number and transmittance as inFIG. 1).

The adducts according to the present invention, when obtained asdescribed, are particularly pure so as to be suitable for pharmaceutcalcompositions intended for general antihaemorrhagic therapy.

In addition to the above, it was found that the addition of menadionebisulfite with other compounds also resulted in substances havingexcellent stability. This was particularly evident in compounds withvitaminic activity having a quaternizable nitrogen atom. These compoundscan be grouped according to the following basic structures:

    --N═C--                                                (1)

The nitrogen atom in (1) may be part of a simple heterocyclic structurehaving one or more nitrogen atoms, for example, a penta- or hexa-atomicstructure, or a complex structure comprising two or more condensedrings, which may be penta- or hexa-atomic aromatic or heterocyclicincluding N, S, O atoms; thus, for example, N may be part of a basicpyridinic ring structure ##STR1## as in nicotine amide or nicotinicacid; a basic thiazolic ring structure ##STR2## as in thiaminehydrochloride (vitamin B₁); and a purinic ring structure ##STR3## as inadenine hydrochloride (vitamin B₄); or ##STR4## wherein --N is thequaternizable nitrogen atom. The sequence of carbon atoms in (2) formspart of an aliphatic or aromatic structure --R₁, R₂, R₃ and R₄independently represent H, an aliphatic or aromatic radical, or afunctional group.

Compounds of structure (2) would include, for example, aminobenzoicacids, particularly p-aminobenzoic acid, aliphatic and aromatic amines,for example, dibenzylethylenediamine. It would also include aminoacids,such as tryptophan(1-alpha-amino-3 indole propionic acid) orhistidine(alpha-amino-4(or 5)-imidazole-propionic acid)(C₆ H₉ N₃ O₂)##STR5##

Another way of describing the adducts of the present invention is KV₁(V₂), wherein K is the K-vitamin compound, V₁ is the stabilizing andtherapeutically active component selected from the first group (1),e.g., nicotinic acid, nicotinic amide, vitamin B₁ and vitamin B₄ ; andV₂ is selected from the second group, e.g., aminobenzoic acid,histidine, and tryptophan. All the compounds (V₁, V₂) share thetherapeutic activity of the new stabilized adducts of the invention.

For a better illustration of the present invention, the followingexamples are provided. These examples are illustrative of the presentinvention and should not be considered as limiting the same.

EXAMPLE I

A solution comprising 18.5 g nicotine amide and 150 ml 1N HCl was mixedwith a solution of 50 g menadione sodium bisulfite in 100 ml of water atroom temperature for about one hour. A white precipitate was obtainedwhich, after filtering, washing and vacuum drying at 50°-60° C., weighed48 g.

The product obtained is slightly soluble in water, m.p. 182°-183° C.with decomposition, and does not contain more than 1% water, asdetermined according to Karl Fischer's method, and its elementaryanalysis is as follows:

Actual: C=54.1%; H=4.1%; N=7.2%; S=8.2%

Theoretical for C₁₇ H₁₆ O₆ N₂ S: C=54.19%; H=4.25%;

N=7.43%; S=8.52%

The percentage of menadione was 45.0%.

EXAMPLE II

A solution comprising 27 g of nicotine amide and 22 g of concentratedHCl (36%) in 120 ml water was admixed at room temperature with asolution comprising 25 g menadione sodium bisulfite in 75 ml water forabout one hour. A precipitate of white color was obtained which, afterfiltering, washing and vacuum drying at 60° C., weighed 27 g.

The percentage of menadione was 45.2%. FIG. 1 shows the IR spectrum ofthis product, referred to as menadione nicotine amide bisulfite (MNB).

EXAMPLE III

A solution comprising 18.5 g nicotinic acid, 18 g concentrated HCl and150 ml water was mixed at room temperature with an aqueous solution ofmenadione sodium bisulfite (50 g in 100 ml water). A white precipitatewas obtained which, after filtration, washing and vacuum drying, weighed47 g.

The product is slightly soluble in water; m.p. 184°-186° C. withdecomposition; it is free of water of crystallization and contains 44.6%by weight of menadione and 33.5% by weight of nicotinic acid. FIG. 2shows the IR spectrum of this product, referred to as menadionenicotinic acid bisulfite (MANB).

EXAMPLE IV

Menadione-bisulfite-p-amino benzoic acid adduct.

    ______________________________________                                        Formula:    C.sub.18 H.sub.17 O.sub.7 N S                                                            M.W. = 391.4                                           ______________________________________                                    

10 g of p-amino benzoic acid were dissolved in 65 g of an 8.5% aqueoussolution of hydrochloric acid. To this solution, 25 g of powderymenadione sodium bisulfite was added. After this addition, the solutionwas cooled to 5° C.; a white precipitate was obtained, which, afterfiltering, washing and vacuum drying, weighed 24 g.

    ______________________________________                                        Analysis: menadione      42.5%                                                          p-aminobenzoic acid                                                                          35.1%                                                          H.sub.2 O       <1%                                                 Water solubility     5.5 g/100 ml at 25° C.                            pH (saturated solution)                                                                            1.8                                                      ______________________________________                                    

FIG. 3 shows the IR spectrum of this product.

EXAMPLE V

Menadione bisulfite-thiamine adduct.

    ______________________________________                                        Formula:   C.sub.23 H.sub.27 Cl N.sub.4 O.sub.6 S.sub.2                                               M.W. = 555.17                                         ______________________________________                                    

25 g of thiamine hydrochloride (vitamin B₁) was dissolved in 50 g ofwater; to said solution of 25 g of powdery sodium menadione bisulfitewas added within about half an hour. After half an hour of agitation,the reaction mass became opalescent and the adduct began to precipitate.After an hour, the precipitate was filtered, washed and dried. 25 g of awhite product having the following analysis was obtained.

    ______________________________________                                        Analysis:   menadione   32.0%                                                             vitamin B.sub.1                                                                           52.1%                                                             H.sub.2 O   <1.0%                                                 Water solubility    6.0 g/100 at 25° C.                                pH (saturated solution)                                                                           2.7                                                       ______________________________________                                    

FIG. 4 shows the IR spectrum of this product.

EXAMPLE VI

Menadione bisulfite adenine adduct.

    ______________________________________                                        Formula:    C.sub.16 H.sub.15 N.sub.5 O.sub.5 S                                                      M.W. = 389.42                                          ______________________________________                                    

10 g adenine was dissolved in a solution comprising 300 g of water and 8g of concentrated 37% hydrochloric acid. To this solution, 25 g ofpowdery menadione sodium bisulfite was added. A white precipitate formedimmediately. The slurry was filtered and washed. 29 g of a whitegranulated product having the following analysis was obtained.

    ______________________________________                                        Analysis:   menadione   43.2%                                                             adenine     34.5%                                                             H.sub.2 O     0%                                                  Water solubility    0.7 g/100 ml at 25° C.                             pH (saturated solution)                                                                           3.3                                                       ______________________________________                                    

FIG. 5 shows the IR spectrum of this product.

EXAMPLE VII

Menadione bisulfite tryptophan adduct.

    ______________________________________                                        Formula:    C.sub.22 H.sub.22 N.sub.2 O.sub.7 S                                                      M.W. = 458.51                                          ______________________________________                                    

15 g of tryptophan was dissolved in 70 g of a solution of 5%hydrochloric acid. To the resulting solution, 25 g of powdery menadionesodium bisulfite was added. The solution remained clear (under stirring)for about 1 hour. Afterwards, a white precipitate started forming; theprecipitate, after filtering, washing and vacuum drying, weighed 25 g.

    ______________________________________                                        Analysis:   menadione   36.5%                                                             tryptophan  44.2%                                                             H.sub.2 O   <1.0%                                                 Water solubility    1.7 g/100 ml at 25° C.                             pH (saturated solution)                                                                           2.1                                                       ______________________________________                                    

FIG. 6 shows the IR spectrum of this product.

EXAMPLE VIII

Menadione bisulfite-histidine adduct.

    ______________________________________                                        Formula:    C.sub.17 H.sub.19 N.sub.3 O.sub.7 S                                                      M.W. = 409.45                                          ______________________________________                                    

16 g of histidine hydrochloride was dissolved in 50 g of a 6% solutionof hydrochloric acid. To this solution, 25 g of powdery menadione sodiumbisulfite was added within about 1/2 hour. A white, very fineprecipitate was obtained, which, after filtering, washing and vacuumdrying, weighed 26 g and had the following analysis:

    ______________________________________                                        Analysis:   menadione   41.2%                                                             histidine   36.5%                                                             H.sub.2 O   <1.0%                                                 Water solubility    1.6 g/100 ml at 25° C.                             pH (saturated solution)                                                                           2.1                                                       ______________________________________                                    

FIG. 7 shows the IR spectrum of this product.

EXAMPLE IX Stabilization Test of Adduct With Nicotine Amide

1.5 g menadione bisulfite nicotine amide adduct, as prepared accordingto Example I, was mixed with 50 g aluminum silicate, containing 11.25%moisture, determined according to Karl Fischer's method. The compoundwas placed in a sealed container and thermostatically maintained at 55°C. for 3 days. After this period, the percentage of menadione wasdetermined according to the method described in USA Pharmacopeia. 75% ofthe original amount of menadione was still present.

EXAMPLE X Stabilization Test Of Adducts With Nicotinic Acid

1.5 g adduct menadione nicotinic acid bisulfite (MANB) preparedaccording to Example III was mixed with 50 g aluminum silicatecontaining 11.25% moisture. The compound was placed in a sealedcontainer and thermostatically maintained at 55° C. for 3 days. Afterthis period, the percentage of menadione was determined according to themethod of USA Pharmacopeia. 86% of the original amount of menadione wasstill present.

At the same time, tests were carried out under the same conditions, butusing vitamin K₃ (menadione sodium bisulfite) and menadione bisulfite2-hydroxy-4,6-dimethylpyrimidine adduct; in the former case, only 15.5%of the originally present menadione was unaltered, whereas in the lattercase, the amount of unaltered menadione was 80.2%.

EXAMPLE XI Stabilization Test Of Adduct With p-aminobenzoic acid

1.5 g menadione bisulfite p-aminobenzoic acid adduct as preparedaccording to Example IV was mixed with 50 g aluminum silicate,containing 11.25% moisture, determined according to Karl Fischer'smethod. The compound was placed in a sealed container andthermostatically maintained at 55° C. for 3 days. After this period, thepercentage of menadione was determined according to the method describedin USA Pharmacopeia. 77% of the original amount of menadione was stillpresent.

EXAMPLE XII Stabilization Test of Adduct With Thiamine

1.5 g menadione bisulfite Thiamine adduct as prepared according toExample V was mixed with 50 g aluminum silicate, containing 11.25%moisture, determined according to Karl Fischer's method. The compoundwas placed in a sealed container and thermostatically maintained at 55°C. for 3 days. After this period, the percentage of menadione wasdetermined according to the method described in USA Pharmacopeia. 77% ofthe original amount of menadione was still present.

EXAMPLE XIII Stabilization Test of Adduct With Adenine

1.5 g menadione bisulfite adenine adduct as prepared according toExample VI was mixed with 50 g aluminum silicate, containing 11.25%moisture, determined according to Karl Fischer's method. The compoundwas placed in a sealed container and thermostatically maintained at 55°C. for 3 days. After this period, the percentage of menadione wasdetermined according to the method described in USA Pharmacopeia. 95% ofthe original amount of menadione was still present.

EXAMPLE XIV Stabilization Test of Adduct With Trypotophan

1.5 g menadione bisulfite tryptophan adduct, as prepared according toExample VII was mixed with 50 g aluminum silicate, containing 11.25%moisture, determined according to Karl Fischer's method. The compoundwas placed in a sealed container and thermostatically maintained at 55°C. for 3 days. After this period, the percentage of menadione wasdetermined according to the method described in USA Pharmacopeia. 85% ofthe original amount of menadione was still present.

EXAMPLE XV Stabilization Test of Adduct With Histidine

1.5 g menadione bisulfite Histidine adduct, as prepared according toExample VIII was mixed with 50 g aluminum silicate, containing 11.25%moisture, determined according to Karl Fischer's method. The compoundwas placed in a sealed container and thermostatically maintained at 55°C. for 3 days. After this period, the percentage of menadione wasdetermined according to the method described in USA Pharmacopeia. 63.5%of the original amount of menadione was still present.

EXAMPLE XVI Preparation of Feeds

When preparing feeds, it is the standard practice to operate under dryand cold conditions. First, one prepares an active principle concentrate(a), which will be incorporated in the composition hereinafter shown at(b), which is a carrier for a feed integrator, thus obtaining a standardintegrator. The standard integrator is diluted at time of use with thefeeds suitable for each animal in the proper proportion.

Thus, for example, a typical integrator compound for feeds will contain,along with the carrier, the following composition of active ingredients.

    ______________________________________                                        (a) Concentrate of active ingredients                                         for kg of integrator                                                                          (a.sub.2) Active concentrate                                  (a.sub.1) Commercial active                                                                   containing the adduct                                         concentrate without adduct                                                                    of the present invention                                      ______________________________________                                        Vitamin A                                                                              10.sup.6 I.U.                                                                            Vitamin A     3,500,000 I.U.                              Vitamin D.sub.3                                                                        200,000 I.U.                                                                             Vitamin D.sub.3                                                                             400,000 I.U.                                Vitamin B.sub.2                                                                        1200 mg    Vitamin E     3,500 mg                                    Vitamin B.sub.6                                                                        600 mg                                                               Vitamin B.sub.12                                                                       1 mg       Vitamin B.sub.2                                                                             400 mg                                      Vitamin PP                                                                             2000 mg    Vitamin B.sub.12                                                                            2 mg                                        Menadione                                                                              2000 mg    Vitamin B.sub.6                                                                             600 mg                                      sodium              Adduct of the 4000 mg                                     bisulphite          invention                                                 (Vitamin K.sub.3)   (menadione nicotine                                                           amide                                                                         bisulfite)                                                (b) Carrier for feed integrator                                               Maize flour            400 g/kg                                               Soia bean flour        200 g/kg                                               Rice flour             100 g/kg                                               Purple medick flour    100 g/kg                                               Peanut flour            50 g/kg                                               Meat flour              50 g/kg                                               Beet molasse            25 g/kg                                               Barley flour            25 g/kg                                               Dicalcium phosphate     25 g/kg                                               Milled CaCO.sub.3       15 g/kg                                               NaCl                    10 g/kg                                               ______________________________________                                    

Then, amino-acids and mineral salts are added thereto.

The integrator thus obtained is in turn diluted in use with a specificfeed suitable for the individual animals to which it is administeredrespectively.

As evident from the foregoing, by replacing the commercial concentrate(a₁) with a concentrate of active ingredients containing the adductaccording to the present invention, the unstable vitamin K₃ and vitaminPP are replaced by a single stable product having the activity of bothof said vitamins. This is clearly an advantage.

EXAMPLE XVII

Another example in which a compound according to the present inventionis used in integrators for standard feeds:

    ______________________________________                                        Vitamin A            1,000,000                                                                              I.U.                                            Vitamin D.sub.3      200,000  I.U.                                            Vitamin B.sub.2      1,200    mg                                              Vitamin B.sub.12     1        mg                                              Vitamin B.sub.6      600      mg                                              Menadione nicotine   1,000    mg                                              amide bisulfite                                                               ______________________________________                                    

EXAMPLE XVIII

This example contemplates the product according to the invention in aconcentrate for a treated integrator, that is, one provided with aspecific therapeutic activity.

The composition of active concentrate for 1 kg of integrator correspondsto:

    ______________________________________                                        Vitamin A            3,000,000                                                                              I.U.                                            Vitamin E            2,000    mg                                              Vitamin B.sub.2      600      mg                                              Vitamin B.sub.12     2        mg                                              Adduct according     8,000    mg                                              to the invention                                                              ______________________________________                                    

At the time of use, this kg of integrator is mixed with 100 kg standardfeed.

What is claimed is:
 1. Adducts of menadione bisulfite withp-aminobenzoic acid having the general formula: C₁₈ H₁₇ O₇ NS, m.w.391.4; menadione 42.5% by weight; p-aminobenzoic acid 35.1% by weight.2. Adducts of menadione bisulfite with adenine having the generalformula: C₁₆ H₁₅ N₅ O₅ S, m.w. 389.42; menadione 43.2% by weight;adenine 34.5% by weight.